Detection and molecular characterization of Avipoxvirus in Culex spp. (Culicidae) captured in domestic areas in Rio de Janeiro, Brazil

Avian pox is a highly contagious poultry disease that causes significant economic losses. Mosquitoes belonging to the genus Culex (Diptera: Culicidae) have a fundamental role in disseminating Avipoxvirus (Poxviridae). This study proposes investigating the presence of Avipoxvirus (APV) DNA in Culex spp. from Rio de Janeiro to determine its frequency and perform a phylogenetic analysis based on the core like the 4b protein (p4b) gene. The detection of APVs was conducted individually on four hundred Culex spp. mosquitoes. A total of 12.23% (47/384) of the Culex spp. were positive in the PCR. Sequencing the p4b gene revealed that this study’s sequences displayed 98.8–99% identity with Fowlpoxvirus (FWPW) sequences available in GenBank. In the phylogenetic analysis, these APVs were clustered in the A1 subclade together with FWPW sequences from several countries. The evolutionary distance of the p4b gene was 0.61 ± 0.21% in rural areas and 0.38 ± 0.16% in peri-urban areas. The current investigation is the first study to report the detection of APVs in field-caught mosquitoes. Moreover, a high frequency of APV DNA was observed in Culex spp. captured in domestic areas, where backyard poultry is present. This data demonstrates the importance of implementing control measures for Culex spp. to mitigate the transmission of APVs in backyard poultry in Rio de Janeiro.

www.nature.com/scientificreports/ APV diversity is primarily classified into three phylogenetical groups (A, B, C), characterized by Fowlpoxvirus (FWPW) (clade A; subdivisions A1-A7), Canarypoxvirus (clade B; subdivisions B1-B3), and Psittacinepoxvirus (clade C) 20 . However, two other clades (D, E) have been proposed 21,22 . Clade D includes a unique APV strain, QP-241, isolated from a Japanese quail collected in Italy 21 . The sequences included in clade E were earlier recorded from outbreaks in turkey herds in Hungary 22 and layer chickens in Mozambique 23 . Ribeiro et al. 24 also reported fowls from the Southern region of Brazil presenting APV sequences clustering in clade E. However, no investigation as to the viral agent in mosquitoes has been performed in Brazil. Moreover, few studies have targeted the circulation of APVs and their identification in mosquito species with vectoring capacities, such as mosquitoes of the Culex genus. Furthermore, it presents an analysis of the phylogenetic relationship and the genetic variability of APVs from Culex spp. collected in rural and peri-urban areas nearby backyard poultry located in the municipality Seropedica, Rio de Janeiro, Brazil.

Results
The PCR applied in the current study, which targets APVs, presented a detection limit of 100 copies of the APV p4b gene fragment. The molecular screening resulted in a frequency of 12.23% (47/384) in Culex mosquitoes. Of these positive samples, 13.63% (21/154) were from peri-urban areas, and 11.30% (26/230) were from rural areas (Fig. 1). There was no significant statistical association between the frequency of APVs in Culex mosquitoes and the analyzed areas (p > 0.05). However, the kernel map identified a hot zone, demonstrating a higher concentration of APV-positive mosquitoes in rural areas of the municipality of Seropedica (Fig. 2).
The qPCR targeting the chicken mitochondrial gene revealed that 91.49% (43/47) of the p4b-positive APV samples were also positive for chicken blood, and 8.51% (4/47) of the p4b-positive APV samples were negative for chicken blood.
A total of ten positive samples were sequenced for phylogenetic reconstruction based on the pb4 gene. The identity percentage of the sequences in this study ranged from 98.8 to 99%, with FWPW sequences available in the GenBank database. All sequences recovered from Culex mosquitoes in this study were clustered within clade A, subclade A1, and denoted as part of the FWPW category. In the same group, the A1 subclade isolates were recovered in Gallus gallus from Brazil, Nigeria, Portugal, and Singapore; Meleagris gallopavo from Iran and Brazil; and Dendrocygna viduata from Brazil (Fig. 3). The global evolutionary distance of the FWPW p4b gene  www.nature.com/scientificreports/ was 0.48 ± 0.14% in the targeted region. The evolutionary distance between FWPW sequences from mosquitoes collected in rural and peri-urban areas was 0.47 ± 0.13%. The evolutionary distance was compared within each evaluated area, obtaining a value of 0.61 ± 0.21% in the rural area and 0.38 ± 0.16% in the peri-urban area (Table 1).

Discussion
Brazil is the second leader in the world's poultry production and the first leader in chicken exportation 25 . Therefore, any negative impact on poultry health can result in substantial commercial losses. In this context, it is of utmost interest to investigate all aspects of poultry health. Avian pox is a viral disease with a high incidence in tropical and subtropical countries 1 . Avipoxviruses have no predilections in terms of host species, sex, or age, targeting more than 374 domestic and wild bird species and making it difficult to achieve environmental eradication 3 . Furthermore, arthropods perform APV transmission, which further exacerbates the potential of dispersion. Mosquitoes are one of many arthropods playing the role of mechanical vector in the Avian pox epidemiological chain, which is a potent threat given that mosquitoes populations in the tropics are high 26 .
Considering the substantial economic loss caused by Avian pox, it is vital to elucidate the transmission chain of APVs by identifying potential transmission sources 5 . Although investigations targeting APVs in mosquitoes are scarce, prior studies have demonstrated that APV detection in potential vectors is possible and can become an essential tool for monitoring the pathogen circulation in areas where the disease occurrence may increase due to climatic factors. In addition, APV detection in potential vectors can further clarify the mosquitoes' participation in the APV transmission chain in a determined area, which could improve the strategic measures to control the Avian pox disease. Furthermore, the present study can contribute to the clarification of the biology of the vectors involved in and strategic measures for vector control, which is of veterinary interest 5,27 .
The frequency of APVs found in field-caught Culex spp. collected in the present investigation (12.23%; 47/384) was higher than previously recorded by Yeo et al. 5 , who observed a positivity of 2.60% (4/154) in mosquito pools collected in areas where outbreaks of Avian pox occurred in Singapore. This divergence may be related to several factors, such as differences in the sampling approaches adopted in these studies, sample conservancy, and the chosen collection sites. Although the studies by Yeo et al. 5 and Lee et al. 12 chose the PCR method for the detection of Avipoxvirus in mosquito and biting midge pools, respectively, the present study applied a distinct sampling method by individually testing Culex specimens. Individual sampling proved to be sufficient to obtain the total amount of DNA after performing the modified extraction technique outlined by Ayres et al. 28 . This  www.nature.com/scientificreports/ height 28 . Moreover, the DNA extracted from the samples obtained in the current investigation was preserved in a DNA stabilization solution and maintained in an ultra-freezer at − 80 °C until molecular detection. Notably, the selected spots were located in areas where outbreaks of Avian pox had occurred in previous years. However, during the present investigation, no Avian pox case was observed or recorded close to the studied points by the owners or local veterinarians. Despite the absence of the disease's clinical signs in backyard chickens, the frequency of APVs in Culex spp. was relatively high in the target area, raising questions about the involvement of other domestic birds species participating in the maintenance of virus circulation. The presence of four Culex spp. that were positive for APVs and negative for the chicken mitochondrial gene reinforced this hypothesis. Therefore, more studies must be performed to elucidate the transmission chain of APVs in the targeted area.
Prior studies targeting APVs-p4b obtained of avian species from Brazil demonstrate the presence of several APVs lineages of distinct clades circulating throughout the country. Studies conducted in the Southeastern region of Brazil, the same area of the current research, have reported the presence of APV sequences clustered in subclade A1 and isolated from both turkeys and chickens 29,30 . Moreover, in the Northeastern region of Brazil, Braga et al. 31 isolated APVs with lineage clustering in the A1 subclade coming from a Brazilian native duck (Dendrocygna viduata) (Fig. 3). The present phylogenetic reconstruction exposes the lineages of APVs recovered from Culex mosquitoes from the state of Rio de Janeiro, also grouped in the A1 subclade (APVs-A1) (Fig. 3).
Historically, the lineages of APVs from subclade A1 were strongly associated with Galliform hosts (Gallus gallus and Meleagris gallopavo) according to the results published by Gyuranecz et al. 20 , Jarmin et al. 32 , and Manarolla et al. 21 . Nevertheless, recent records of the APVs-A1 sequence infecting a Brazilian native duck (Order Anseriformes) reported by Braga et al. 31 have raised questions about this subclade's specificity within the species of the Galliformes order. Furthermore, given the fact that the most common APV host species of subclade A1 (Gallus and Meleagris gallopavo) are exotic species introduced by humans as food resources in most regions of the globe, such as in Brazil, it is possible to suggest that the lineages of APVs of the A1 subclade accompany their hosts in species introduction around the globe. However, given the record of an APVs-A1 infecting a native Brazilian species 31 , it is possible to suggest a community APVs-A1 transmission between different avian host species.
The species-level identification in Culex spp. requires genitalia dissection, making such samples unviable for molecular analysis. Cytochrome oxidase I (COI) DNA barcoding contains information for identifying mosquitoes of the Culex genus 33 . However, a criticism of using the COI barcode for specification is the ambiguous identification or the absence of clusters in phylogenetic trees of recently diverged species [34][35][36] . Algorithms were developed by Meier et al. 37 and van Velzen et al. 38 to improve COI barcoding identification at the species level. However, Laurito et al. 39 employed a COI barcode and BCM algorithm to identify Culex spp. from Brazil and Argentina and concluded that the COI barcode does not contain enough information to distinguish Culex spp. Thus, the present study could not determine the real prevalence of APVs associated with a particular Culex species. Nevertheless, most Culex species found in peri-urban and rural areas in the state of Rio de Janeiro are highly ecologically similar [40][41][42][43][44][45] . Therefore, the differences do not substantially impact the control measures applied to Culex species and the transmission of APVs.
The present research reports on the genetic diversity of APVs. The most divergent APV sequences were obtained in mosquito samples from rural areas. This result may indicate pathogen adaptation to highly anthropized areas since significant genetic divergence is present in areas with lower anthropization levels due to more significant interactions and an abundance of host species found in these regions. Giraudeau et al. 46 demonstrated for the first time that the highest rates of Avipoxvirus infection in finches (Haemorhous mexicanus) were in urban areas, where human activities were more often present.

Methods
Study area. The present study was conducted in the municipality of Seropedica (22° 44′ 38″ south latitude; 43° 42′ 27″ west longitude) in Rio de Janeiro as represented in the Fig. 1, from June 2016 to July 2017. Mosquito collections were carried out in properties with backyard poultry from rural and peri-urban areas. Table 1. Evolutionary distance (%) between p4b gene sequences of Avipoxvirus obtained from Culex spp. in rural and peri-urban areas in the municipality of Seropedica, Rio de Janeiro, Brazil. www.nature.com/scientificreports/ Mosquito collections and identification of Culex spp. The parameters of an infinite population were considered based on the desired level of prevalence to determine the sample size 47 , with a sample error of 5%, a confidence level of 95%, and an expected prevalence of 50%, resulting in a total of 384 mosquitoes in sampling.
The collections were carried out using CDC light traps in peri-domestic areas, which operated for 12 h, three times a week, totaling 864 h of operation of the traps from July 2016 to July 2017. A total of 2839 mosquitoes of the Anopheles, Aedes, and Culex genera were collected, with the Culex genus being the most abundant and comprising 96.23% (2732/2839). The identification of mosquitoes at the genus level was performed by morphological structures common to the Culex spp. according to Forattini 43 and Berlin and Belkin 48 . Characteristics such as sex and engorgement were considered. The specimens were individually placed in tubes containing RNAlater® solution and stored at -20 °C until molecular analysis was performed.
DNA extraction. DNA extraction was performed for each specimen of Culex spp. Before the extraction protocol, each specimen stored in RNAlater® (ThermoFisher Scientific) had its eyes dissected and discarded to avoid possible inhibitory action in molecular analysis caused by pigmentary components from insect eyes 49 . Each body was washed three times with 500 μL of sterile PBS and centrifuged at 14,500×g for 3 min to remove the RNAlater® solution. A protocol published by Ayres et al. 28 was performed for DNA extraction.  www.nature.com/scientificreports/ sequence was obtained using the BLASTn tool available at https:// blast. ncbi. nlm. nih. gov/. The phylogenetic reconstruction was performed with 61 sequences available in GenBank belonging to subclades A1 to A7, B1 to B3, clades C, D, and E were compared to the samples obtained in this study. Sequences were aligned in MAFFT software using default options, and then the matrix was visually inspected for inconsistencies 53 . After removing misaligned positions with GBlocks, according to Talavera and Castresana 54 , a final matrix was obtained. The best replacement model was determined using JModelTest software 55 . The inference of the Avipoxvirus phylogeny was performed using the Maximum Likelihood (ML) method. The clade bootstrap values were evaluated using the RaxML self-convergence criterion with the best pseudo-replica values 56 .
Georeferencing. The Brazilian state cartographic base was collected in shapefile format from the electronic database of the Brazilian Institute of Geography and Statistics 57 and uploaded in the GIS software [QGIS 3.22.8 'Białowieża' (https:// qgis. org/ pt_ BR/ site/], maintaining the coordinates in degrees in the Geocentric Reference System for the Americas (SIRGAS 2000, previously established by IBGE. Thematic maps were created by applying the geographical coordinates of the collected samples in the cartographic base. A kernel map was also designed to visually distinguish infection clusters through interpolating the Avipoxvirus-positive samples and the study sites. The colored scale in the obtained raster was proportional to the frequency, ranging from 1 to 9. Statistical analysis. The frequency of Avipoxvirus in Culex spp. captured in the municipality of Seropedica in the state of Rio de Janeiro was compared using the Chi-Square (χ 2 ) test, admitting an error of 5% using R software version 3.6.1 58 .

Conclusion
This study reported a high frequency of FWPW in Culex spp. in Seropedica, indicating a risk in implementing an organic system for poultry production in Seropedica, Rio de Janeiro. The FWPW circulation in this area reinforces the importance of vaccination as a preventive measure. In addition, this is the first study to report FWPW sequences obtained from Culex mosquitoes collected in the southwestern region of Rio de Janeiro, Brazil.